Tumor necrosis factor-alpha (TNF-alpha) is a protein that is an essential mediator of the inflammatory response. TNF-alpha is identical to the hormone cachectin, which is a key endogenous factor in the pathogenesis of chronic wasting associated with acute inflammatory or malignant diseases. B. Beutler and A. Cerami, "Tumor Necrosis, Cachexia, Shock, and Inflammation: A Common Mediator", Ann. Rev. Biochem., 57:505-518 (1988).
TNF-alpha has antitumor activity, causing hemorrhagic necrosis in tumors in mice treated with bacillus Calmette-Guerin (BCG) and endotoxin. TNF-alpha is the major mediator of endotoxin shock (Science 229:869 (1985)) in which hypotension, derangement of lipid and glucose metabolism, acidosis, and widespread neutrophil activation can lead to increased catabolic metabolism and possible death of the organism. Ann. Rev. Biochem. 57:505 (1988).
Cachexia, i.e., anorexia and wasting in chronic infectious or malignant diseases, is also evoked by TNF-alpha primarily through suppression of the enzyme lipoprotein lipase and by mobilization of triglycerides in adipocytes. Mol. Biochem. Parasitol., 2:31 (1980). Through its complex effect on endothelial and neutrophil cells, TNF-alpha also evokes neutrophil adhesion to lung capillaries and enhances thrombus formation. This may result in disseminated intravascular coagulation, migratory thrombosis and hemorrhagic necrosis. TNF-alpha is a potent endogenous pyrogen affecting hypothalmic neurons and promoting interleukin-1 (IL-1) production. TNF has also been implicated in the pathogenesis of malaria and gram-negative septic shock. See Ann. Rev. Biochem. 57:505-518 (1988) supra and references cited therein.
TNF-alpha monomer is a polypeptide of 17 kd molecular weight consisting of 150-160 amino acids. B. Beutler and A. Cerami, Nature 320:584-588 (1986). The protein can be isolated from the supernatant of stimulated human macrophages, or, in a highly pure form, as a recombinant protein. Shirai, T. et al., Nature, 313:803-806 (1985). The bioactivity of TNF-alpha is indistinguishable from that of the cytotoxic polypeptide hormone lymphotoxin, produced by activated lymphocytes (also identified as TNF-beta). Hybridoma 6(5):489 (1987). The two molecules are structurally related, being encoded by closely linked genes within the major histocompatibility complex. Nucleic Acid Res. 13:6361 (1985); PNAS 83:8699 (1986). As a result, the two proteins share common receptors on a large variety of cells. TNF-alpha and beta can be distinguished from each other based on their antigenic properties but not on the basis of cytotoxic activity.
TNF-alpha is produced by all types of macrophages in a degree dependent on the maturation and differentiation state (J. Immunology 138:957 (1987)), as well as by T lymphocytes (J. Exp. Med. 165:1581 (1987)) in response to a variety of invasive stimuli. The most potent known stimulus is the bacterial lipopolysaccharide (LPS). Viruses, trypanosoma, plasmodium, some Gram-positive bacteria, and the lymphokine IL-1 also stimulate TNF-alpha production. Beutler and Carami, supra.
TNF-alpha is, however, subject to rapid physiochemical changes in vitro and this is likely to result in lower measured TNF values in immunoassays as compared in in vivo concentrations. TNF-alpha dissociates and aggregates to yield a protein of variable size. The biologically active form of TNF-alpha is believed to be a dimeric or trimeric species. Smith, R. A. and C. Baglioni, J. Biol. Chem. 262:6951-6954 (1987); Davis, J. M. et al., Biochemistry, 26:1322-1326. Dissociation of the trimeric protein is believed to be responsible for the instability of biologically active TNF-alpha in dilute solutions. Smith and Baglioni, Id. A failure to detect TNF-alpha activity in stored sera has also been reported. Blood 68:337 (1986); Blood 64:229 (1984).
Immunoassays for TNF-alpha have been described in the literature. For example, EPO Published Application 218,868 discloses the preparation of pure human tumor necrosis factor. The application further discloses hybridomas which produce monoclonal antibodies to human tumor necrosis factor and use of the monoclonal antibodies for diagnostic purposes. JP Published Application 60208924 discloses a monoclonal antibody to human tumor necrosis factor and its use in immunoassays.
Because the degradation of TNF-alpha in fresh, blood-derived materials such as sera or plasma can occur immediately after the biological sample is removed from the body, there has been some interest in developing a method to preserve TNF activity. Two recent U.S. Pat. Nos. 4,447,355 and 4,457,916, are directed to methods of stabilizing the activity of frozen or lyopholized TNF using albumin or a carbohydrate material. Unfortunately, neither of these methods addresses the question of preserving biologically active TNF-alpha preparations for in vitro use.
Present methods that measure TNF-alpha concentrations in biological samples (e.g., immunometric or bio-assays) give result that merely reflect TNF-alpha activity after degradation, and do not accurately measure activity relevant to the situation in vivo. A method capable of preventing the accelerated degradation of TNF-alpha in fresh samples will provide a valuable tool for more accurate TNF-alpha measurements. Such a method will be useful in diagnosis and management of a variety of pathological states, infectious diseases such as gram-negative septic shock or malignant diseases.